EP0906575A1 - Marker für männerunfruchtbarkeit und/oder -fruchtbarkeit - Google Patents

Marker für männerunfruchtbarkeit und/oder -fruchtbarkeit

Info

Publication number
EP0906575A1
EP0906575A1 EP97915240A EP97915240A EP0906575A1 EP 0906575 A1 EP0906575 A1 EP 0906575A1 EP 97915240 A EP97915240 A EP 97915240A EP 97915240 A EP97915240 A EP 97915240A EP 0906575 A1 EP0906575 A1 EP 0906575A1
Authority
EP
European Patent Office
Prior art keywords
sperm
fertile
spermatozoa
antibody
related antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP97915240A
Other languages
English (en)
French (fr)
Other versions
EP0906575B1 (de
Inventor
Robert Sullivan
Franck Boue
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immucon Inc
Original Assignee
Immucon Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immucon Inc filed Critical Immucon Inc
Publication of EP0906575A1 publication Critical patent/EP0906575A1/de
Application granted granted Critical
Publication of EP0906575B1 publication Critical patent/EP0906575B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/806—Fertility tests
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces
    • Y10S435/906—Streptomyces venezuelae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the invention relates to a marker for male infertility and/or fertility and to a method for the diagnosis of male infertility.
  • Infertility occurs in approximately 8% of North American couples. In 50% of these cases, male factors contribute to this pathological situation. Standard semen analysis including sperm concentration, motility, and morphology are widely used as principal indicators of male fertility. However, these parameters fail to explain all cases of male infertility and do not pro ⁇ vide a precise prognostic value of human fertility in vi vo or in vi tro . Due to the fact that standard semen parameters often provide a poor prognostic value, many other laboratory tests have been developed in order to evaluate the fertilizing ability of human spermatozoa.
  • sperm membrane integrity by hypoosmotic swelling test, nuclear matur ⁇ ity, acrosomal status, motility assessment by video- imaging as well as the ability of sperm to interact with the oocyte.
  • This sperm function has been evalu ⁇ ated by the zona-free hamster test and by the ability of human spermatozoa to bind to homologous zonae pellu- cidae.
  • Therapeutic in vi tro fertilization represents a direct evaluation of the fertilizing ability of semen. The prognostic value of the various andrology tests can thus be determined by correlating their conclusions with in vi tro fertilization results.
  • sperm-zona pellucida binding may be associated with certain cases of male infertility. Therefore, laboratory tests that evaluate the ability of sperm to undergo successfully this step in gamete interaction could have clinical usefulness in the diagnosis of male infertility. Using different animal models, it has been shown that during epididymal transit the male gamete acquires its fertilizing ability. During this matura- tional process, sperm surface proteins are added or modified and these modifications are involved in the acquisition of the ability to the spermatozoa to bind to the zona pellucida.
  • P34H human epididymal sperm protein
  • P34H appears to be involved in one of the absolute prerequisites of fer ⁇ tilization, i.e. sperm-zona pellucida binding. It would be highly desirable to be provided with a marker for male infertility and/or fertility.
  • One aim of the present invention is to provide a marker for male infertility and/or fertility.
  • Another aim of the present invention is to pro ⁇ vide for a method for the diagnosis of male infertil- ity.
  • a method for the diagnosis of male infertility which comprises the steps of: a) determining the amount of P34H or a related antigen thereof in a sperm sample; and b) comparing the determined amount of step a) with a positive control having an amount of P34H or a related antigen thereof of the levels of fertile samples, wherein a P34H or a related antigen value lower than 30% relative to the positive control sample is indicative of infertility.
  • the amount of P34H or a related antigen in step a) may be determined using an antibody raised against one member selected from the group consisting of P34H, a related antigen thereof and fragments thereof. Most preferably, the antibody is raised against P34H.
  • the antibody may be a polyclonal or a monoclonal antibody.
  • kits for the diagnosis of male infertility which comprises: a) an antibody raised against one member selected from the group consisting of P34H, a related antigen thereof and fragments thereof; b) a reagent for detecting the antibody of a) linked to P34H or a related antigen thereof; and c) a positive control having an amount of P34H or a related antigen thereof of the levels of fertile samples.
  • the antibody is enzyme-labeled and the reagent is an enzyme substrate.
  • the reagent is a radiolabeled P34H.
  • the positive control is a fertile sample.
  • the following terms are defined below.
  • P34H or a related antigen thereof refers to the protein P34H or a protein with an homology of about
  • the method and the kit of the present invention may be used to determine male infertility of man and animals which express P34H or a related antigen thereof.
  • the common characteristic of all the male tested in accordance with the present invention is the present of P34H or a related antigen thereof in their sperm sample.
  • Figs. IA and IB illustrate silver stained
  • Fig. 2 is an analysis of amounts of P34H as determined by densitometric scan of immunoblots of pro ⁇ teins extracted from 5.10 ⁇ spermatozoa from fertile (o) and infertile (•) men;
  • Figs. 3A and 3B illustrate silver stained (Fig. 3A) and corresponding immunoblots (Fig. 3B) probed with anti-P34H antiserum of SDS-PAGE of proteins extracted from 5.10 ⁇ human spermatozoa from fertile men before (1) or after a discontinuous Percoll centrifuga ⁇ tion (2,3); and
  • Figs. 4A-4C are Western blot analyses of the number of spermatozoa from control fertile donor (X axis) or from different men (Y axis) bound per human zona pellucida.
  • sperm-zona pellucida binding assay was performed with spermatozoa from fertile and infertile men. Individual men have similar P34H levels on their spermatozoa obtained from different semen samples. However, the amount of P34 varied from one man to another. Compared to a popula ⁇ tion of fertile men, 9/16 infertile men had a P34H level of less than 30% of the normal value, while the remaining 7 males were in the normal range.
  • Sperm from infertile subjects with a normal P34H determination bound to the zonae pellucidae as efficiently as fertile controls.
  • Infertility was defined by a period of at least 30 months of unpro- tected sexual intercourse. In all cases, infertility was classified as idiopathic based on a full infertil ⁇ ity work-up. In women, clinical investigation included; negative hysterosalpingography and laparo- scopy, normal ovulation as evaluated by basal body tem- perature, plasma hormone concentrations, normal endometrial biopsy and post-coital test. The men had produced at least three normal spermiograms as defined by the WHO criteria and were free from anti-sperm anti ⁇ bodies as determined by a negative immunobead test.
  • Proteins from 5.10 ⁇ spermatozoa from each semen sample were submitted to electrophoresis in presence of sodium dodecyl sulfate on a 12% acrylamide gel. The gels were stained with silver nitrate or electro-trans ⁇ ferred to nitrocellulose membranes. These Western blots were saturated for two hours in PBS containing 10% defatted milk and 2% normal goat serum followed by an overnight incubation with the anti-P34H antiserum diluted 1:1000 in PBS supplemented with 1% goat serum. Three volumes of anti-P34H polyclonal antiserum was first adsorbed overnight on one volume of powder of human keratin.
  • Human sperm-zona pellucida binding Human sperm-zona pellucida binding assays were performed with spermatozoa labeled with two different fluorochromes .
  • human oocytes obtained in the context of therapeutic in vi tro fertilization and that had remained unfertilized after 48-60 h post insemination were stored in liqui nitrogen. Immedi ⁇ ately prior to use, oocytes were cashed by transfer into five successive baths of D-PBS containing 0.5% BSA (bovine serum albumin, fraction V) followed by a final wash in B2 medium (Bio Merieux, Marcy-L'Etoile, France).
  • BSA bovine serum albumin
  • Semen samples were submitted to a discon- tinuous Percoll gradient as described above.
  • Motile spermatozoa recovered in the pellet under the 90% Per ⁇ coll layer, were capacitated in B2 medium at 37°C in 5% C ⁇ 2 for 3 hours.
  • Spermatozoa prepared from semen of different men were labeled with one of the two fluoro- chromes FITC (fluorescein) or TRITC (tetramethylrhodamine isothiocyanate).
  • FITC fluorescein
  • TRITC tetramethylrhodamine isothiocyanate
  • spermatozoa were washed and resuspended in B2 medium at a final concentration of 2.10- ⁇ motile spermatozoa/ml.
  • Equal volumes of FITC and TRITC labeled spermatozoa were mixed and incubated at 37 °C in 5% CO2 with human zonae pellucidae.
  • the zonae pelluci- dae were extensively washed by successive pipetting and individually observed under an epifluorescence micro ⁇ scope at the appropriate wavelength corresponding to FITC and TRITC in order to determine the number of spermatozoa from each category that bound to the zona pellucida.
  • each zona pellucida was inseminated with sperm from a fertile donor along with the same number of sperm from a second fertile donor (Fig. 4A), as well as with sperm obtained from infertile men with a normal level of P34H (Fig. 4B) or infertile men with low levels of P34H (Fig. 4C).
  • the Y axis corresponds to fertile (A), infertile men with (B) or without (C) P34H.
  • Each value corresponds to one zona pellucida.
  • the regression line corresponding to each panel is illustrated.
  • P34H is localized on the surface covering the acrosomal cap of ejaculated human spermatozoa (Boue F. et al., 1994, Biol . Reprod. , 51:577-587); a localiza ⁇ tion that is in agreement with the proposed function. In fact, it is via this subcellular region that the male gamete interacts with the zona pellucida.
  • the amount of P34H found on a constant number of spermato ⁇ zoa was highly variable from one individual to the other (Figs. 1 and 2).
  • the protein is present on spermatozoa of all these fertile men that were investigated while lower amounts of this sperm antigen were encountered only in normospermic infertile men (Fig. 2).
  • this sperm antigen could thus be considered as a biochemical marker of male fertility.
  • the variability of P34H is due to an inter-individual variability and that this parameter is consistent from one semen sample to the other from the same individual (Table 1).
  • the variation of P34H on spermatozoa of different buoyant densities could not explain the important variation of P34H level found in our group of infertile men (Fig. 3).
  • Two populations of idiopathic infertile men can be defined on the basis of P34H levels.
  • P34H is a biochemical marker that could be readily used for the diagnosis of male infertility.
  • infertility is associated with the inabil ⁇ ity of the spermatozoa to interact efficiently with the zona pellucida.
  • the rather high occurrence of this defect in our normospermic infertile man population suggests that dysfunction may be a common cause of infertility.
  • vi tro fertilization is presently applied to overcome certain cases of male infertility. Compared to the results obtained when these technolo ⁇ gies are applied to overcome tubal diseases, the chances of successful in vi tro fertilization applied to male infertility is much lower.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Compositions Of Oxide Ceramics (AREA)
  • Transition And Organic Metals Composition Catalysts For Addition Polymerization (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP97915240A 1996-04-18 1997-04-14 Marker für unfruchtbarkeit und/oder -fruchtbarkeit bei männern Expired - Lifetime EP0906575B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US634340 1996-04-18
US08/634,340 US5723305A (en) 1996-04-18 1996-04-18 Marker for male infertility and/or fertility
PCT/CA1997/000249 WO1997040386A1 (en) 1996-04-18 1997-04-14 Marker for male infertility and/or fertility

Publications (2)

Publication Number Publication Date
EP0906575A1 true EP0906575A1 (de) 1999-04-07
EP0906575B1 EP0906575B1 (de) 2004-08-18

Family

ID=24543389

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97915240A Expired - Lifetime EP0906575B1 (de) 1996-04-18 1997-04-14 Marker für unfruchtbarkeit und/oder -fruchtbarkeit bei männern

Country Status (6)

Country Link
US (1) US5723305A (de)
EP (1) EP0906575B1 (de)
AT (1) ATE274187T1 (de)
AU (1) AU2284997A (de)
DE (1) DE69730327D1 (de)
WO (1) WO1997040386A1 (de)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5989549A (en) * 1998-06-08 1999-11-23 Immucon Inc. Acrosomal sperm protein and uses thereof
GB9817795D0 (en) 1998-08-14 1998-10-14 Genosis Inc A method and device for separation and detection of spermatozoa in a sample
US6995252B2 (en) 2000-05-25 2006-02-07 Queen's University At Kingston PT32 sperm protein, sperm c-Yes, oocyte cytoplasmic c-Yes and uses thereof
EP1513865A4 (de) * 2002-05-28 2006-05-03 Univ Minnesota Crisp-polypeptide als verhütungsmittel und inhibitoren der spermienkapazitation
RU2342660C2 (ru) * 2006-11-28 2008-12-27 Государственное образовательное учреждение высшего профессионального образования "Ивановская государственная медицинская академия Федерального агентства по здравоохранению и социальному развитию" Способ прогнозирования нарушений фертильности у мужчин
WO2008142396A1 (en) * 2007-05-18 2008-11-27 Anthony Denis Maher Methods of assessing male fertility using a uridine marker
JP2011515677A (ja) * 2008-03-20 2011-05-19 ザンバー，ティドハー 男性の生殖能診断用の方法及びキット
CN102925478B (zh) * 2012-09-14 2015-05-20 华南农业大学 一种水稻雄性育性可控系的构建方法及育种方法
EP3568700B1 (de) * 2017-01-15 2022-09-28 Arex Life Sciences, LLC Verfahren zur verbesserung der fertilität bei der künstlichen befruchtung
EP3607318A1 (de) 2017-04-06 2020-02-12 Universität zu Köln Verfahren zur bestimmung der fertilität von spermatozoen
WO2020115291A1 (en) 2018-12-06 2020-06-11 Universität Zu Köln Marker for determining the fertility of spermatozoa

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9740386A1 *

Also Published As

Publication number Publication date
DE69730327D1 (de) 2004-09-23
WO1997040386A1 (en) 1997-10-30
ATE274187T1 (de) 2004-09-15
AU2284997A (en) 1997-11-12
US5723305A (en) 1998-03-03
EP0906575B1 (de) 2004-08-18

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